Gene therapy for guanidinoacetate methyltransferase deficiency restores cerebral and myocardial creatine while resolving behavioral abnormalities
2022; Cell Press; Volume: 25; Linguagem: Inglês
10.1016/j.omtm.2022.03.015
ISSN2329-0501
AutoresSuhail Khoja, Jenna Lambert, Matthew Nitzahn, Adam Eliav, Yuchen Zhang, Mikayla Tamboline, Colleen T. Le, Eram Nasser, Yunfeng Li, Puja Patel, Irina Zhuravka, Lindsay M. Lueptow, Ilona Tkachyova, Shili Xu, Itzhak Nissim, Andreas Schulze, Gerald S. Lipshutz,
Tópico(s)Metabolism and Genetic Disorders
ResumoCreatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism. Creatine deficiency disorders are inborn errors of creatine metabolism, an energy homeostasis molecule. One of these, guanidinoacetate N-methyltransferase (GAMT) deficiency, has clinical characteristics that include features of autism, self-mutilation, intellectual disability, and seizures, with approximately 40% having a disorder of movement; failure to thrive can also be a component. Along with low creatine levels, guanidinoacetic acid (GAA) toxicity has been implicated in the pathophysiology of the disorder. Present-day therapy with oral creatine to control GAA lacks efficacy; seizures can persist. Dietary management and pharmacological ornithine treatment are challenging. Using an AAV-based gene therapy approach to express human codon-optimized GAMT in hepatocytes, in situ hybridization, and immunostaining, we demonstrated pan-hepatic GAMT expression. Serial collection of blood demonstrated a marked early and sustained reduction of GAA with normalization of plasma creatine; urinary GAA levels also markedly declined. The terminal time point demonstrated marked improvement in cerebral and myocardial creatine levels. In conjunction with the biochemical findings, treated mice gained weight to nearly match their wild-type littermates, while behavioral studies demonstrated resolution of abnormalities; PET-CT imaging demonstrated improvement in brain metabolism. In conclusion, a gene therapy approach can result in long-term normalization of GAA with increased creatine in guanidinoacetate N-methyltransferase deficiency and at the same time resolves the behavioral phenotype in a murine model of the disorder. These findings have important implications for the development of a new therapy for this abnormality of creatine metabolism. IntroductionCreatine has an essential role in energy homeostasis, being particularly important in muscle and the brain due to their fluctuating energy demands. Outside of the buffering and transport function of high-energy phosphates, creatine is important for neurite growth cone migration, dendritic and axonal elongation, co-transmission on GABA postsynaptic receptors in the central nervous system (CNS)1Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: the 'phosphocreatine circuit' for cellular energy homeostasis.Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1585) Google Scholar, 2Wyss M. Kaddurah-Daouk R. Creatine and creatinine metabolism.Physiol. Rev. 2000; 80: 1107-1213Crossref PubMed Scopus (1873) Google Scholar, 3Almeida L.S. Salomons G.S. Hogenboom F. Jakobs C. Schoffelmeer A.N. Exocytotic release of creatine in rat brain.Synapse. 2006; 60: 118-123Crossref PubMed Scopus (99) Google Scholar, 4Neu A. Neuhoff H. Trube G. Fehr S. Ullrich K. Roeper J. Isbrandt D. 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The creatine kinase system and pleiotropic effects of creatine.Amino Acids. 2011; 40: 1271-1296Crossref PubMed Scopus (428) Google ScholarParallel to dietary consumption, creatine biosynthesis occurs in two enzymatic steps primarily in the liver, kidneys, and pancreas. l-Arginine:glycine amidinotransferase (AGAT) catalyzes the formation of guanidinoacetate (GAA) from arginine and glycine; guanidinoacetate N-methyltransferase (GAMT; EC 2.1.1.2) subsequently catalyzes the formation of creatine by GAA methylation from S-adenosylmethionine.7Sinha A. Ahmed S. George C. Tsagaris M. Naufer A. von Both I. Tkachyova I. van Eede M. Henkelman M. Schulze A. Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.J. Inherit. Metab. Dis. 2020; 43: 827-842Crossref PubMed Scopus (2) Google Scholar Once synthesized, creatine is distributed through the bloodstream and is taken up through the cellular creatine transporter solute carrier family 6 member 8 (SLC6A8), a sodium and chloride-dependent symporter, against a large concentration gradient.8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar Phosphocreatine reversibly transfers its N-phosphoryl group to ADP to regenerate ATP to prevent tissues from running out of energy. As at least half of creatine is synthesized endogenously, deficits in synthesis or transport result in cerebral creatine deficiency syndromes.The cerebral creatine deficiency syndromes include the two autosomal recessive creatine biosynthetic disorders GAMT deficiency8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar,9Stockler S. Marescau B. De Deyn P.P. Trijbels J.M. Hanefeld F. Guanidino compounds in guanidinoacetate methyltransferase deficiency, a new inborn error of creatine synthesis.Metabolism. 1997; 46: 1189-1193Abstract Full Text PDF PubMed Scopus (90) Google Scholar (MIM: 601240) and AGAT deficiency8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar (MIM: 602360). Mutations of SLC6A8 (MIM: 300352) affect creatine transport into cells. The hallmark of this family of disorders is the near-complete absence of creatine in the brain10Schulze A. Bachert P. Schlemmer H. Harting I. Polster T. Salomons G.S. Verhoeven N.M. Jakobs C. Fowler B. Hoffman G.F. et al.Lack of creatine in muscle and brain in an adult with GAMT deficiency.Ann. Neurol. 2003; 53: 248-251Crossref PubMed Scopus (93) Google Scholar and the associated, predominantly neurological, disease. While signs and symptoms can range from mild to severe, intellectual disability, global developmental delay, speech impairment, extrapyramidal movement disorders, autism spectrum disorder, and seizures are common in all three.8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar,11Arias A. Corbella M. Fons C. Sempere A. Garcia-Villoria J. Ormazabal A. Poo P. Pineda M. Vilaseca M.A. Campistol J. et al.Creatine transporter deficiency: prevalence among patients with mental retardation and pitfalls in metabolite screening.Clin. Biochem. 2007; 40: 1328-1331Crossref PubMed Scopus (56) Google Scholar,12Braissant O. Henry H. Beard E. Uldry J. Creatine deficiency syndromes and the importance of creatine synthesis in the brain.Amino Acids. 2011; 40: 1315-1324Crossref PubMed Scopus (92) Google Scholar Together, the creatine deficiency disorders may represent one of the most frequent metabolic disorders with a primarily neurological phenotype7Sinha A. Ahmed S. George C. Tsagaris M. Naufer A. von Both I. Tkachyova I. van Eede M. Henkelman M. Schulze A. Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.J. Inherit. Metab. Dis. 2020; 43: 827-842Crossref PubMed Scopus (2) Google Scholar.Of the creatine deficiency disorders, GAMT loss-of-function mutations tend to result in the most severe phenotype. While likely underdiagnosed,5Hanna-El-Daher L. Beard E. Henry H. Tenenbaum L. Braissant O. Mild guanidinoacetate increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain cell development.Neurobiol. Dis. 2015; 79: 14-27Crossref PubMed Scopus (28) Google Scholar the prevalence is estimated to range from 1 in 114,07213Viau K.S. Ernst S.L. Pasquali M. Botto L.D. Hedlund G. Longo N. Evidence-based treatment of guanidinoacetate methyltransferase (GAMT) deficiency.Mol. Genet. Metab. 2013; 110: 255-262Crossref PubMed Scopus (18) Google Scholar to 1 in 250,000 births,14Mercimek-Mahmutoglu S. Pop A. Kanhai W. Fernandez Ojeda M. Holwerda U. Smith D. Loeber J. Schielen P.C. Salomons A pilot study to estimate incidence of guanidinoacetate methyltransferase deficiency in newborns by direct sequencing of the GAMT gene.Gene. 2016; 575: 127-131Crossref PubMed Scopus (12) Google Scholar with a carrier frequency from 1 in 1,47515Mercimek-Mahmutoglu S. Ndika J. Kanhai W. de Villemeur T.B. Cheillan D. Christensen E. Dorison N. Hannig Y. Hofstede F.C. Lion-Francois L. et al.Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.Hum. Mutat. 2014; 35: 462-469Crossref PubMed Scopus (26) Google Scholar to 1 in 812;16Desroches C.L. Patel J. Wang P. Minassian B. Marshall C.R. Salomons G.S. Mercimek-Mahmutoglu S. Carrier frequency of guanidinoacetate methyltransferase deficiency in the general population by functional characterization of missense variants in the GAMT gene.Mol. Genet. Genomics. 2015; 290: 2163-2171Crossref PubMed Scopus (13) Google Scholar numerous different mutations (missense being the most common) have been reported15Mercimek-Mahmutoglu S. Ndika J. Kanhai W. de Villemeur T.B. Cheillan D. Christensen E. Dorison N. Hannig Y. Hofstede F.C. Lion-Francois L. et al.Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.Hum. Mutat. 2014; 35: 462-469Crossref PubMed Scopus (26) Google Scholar to be scattered throughout the gene, with no hotspot or predominant mutation. The alteration of the Cr/PCr/CK system appears to be of particular importance during early brain development.7Sinha A. Ahmed S. George C. Tsagaris M. Naufer A. von Both I. Tkachyova I. van Eede M. Henkelman M. Schulze A. Magnetic resonance imaging reveals specific anatomical changes in the brain of Agat- and Gamt-mice attributed to creatine depletion and guanidinoacetate alteration.J. Inherit. Metab. Dis. 2020; 43: 827-842Crossref PubMed Scopus (2) Google Scholar Developmental delay is typically detected at 3–12 months;8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar muscular hypotonia, involuntary movements, ataxia, and autistic or self-aggressive behavior are common.8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar,17Schulze A. Ebinger F. Rating D. Mayatepek E. Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation.Mol. Genet. Metab. 2001; 74: 413-419Crossref PubMed Scopus (123) Google Scholar,18Stockler-Ipsiroglu S. van Karnebeek C. Longo N. Korenke G.C. Mercimek-Mahmutoglu S. Marquart I. Barshop B. Grolik C. Schlune A. Angle B. et al.Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring.Mol. Genet. Metab. 2014; 111: 16-25Crossref PubMed Scopus (74) Google Scholar Severe expressive language delay is an almost constant feature;19Vodopiutz J. Item C.B. Hausler M. Korall H. Bodamer O.A. Severe speech delay as the presenting symptom of guanidinoacetate methyltransferase deficiency.J. Child Neurol. 2007; 22: 773-774Crossref PubMed Scopus (18) Google Scholar most patients have no speech or language and, if present, is extremely limited, with marked intellectual disability. Extrapyramidal movements and seizures are characteristic and often refractory to antiepileptics. With deficiency of GAMT, creatine synthesis is markedly impaired, while GAA, accumulating in the plasma, cerebrospinal fluid (CSF), urine, brain, and other tissues, is thought to be the cause of the severe phenotype,8Schulze A. Creatine deficiency syndromes.Mol. Cell Biochem. 2003; 244: 143-150Crossref PubMed Scopus (190) Google Scholar,17Schulze A. Ebinger F. Rating D. Mayatepek E. Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation.Mol. Genet. Metab. 2001; 74: 413-419Crossref PubMed Scopus (123) Google Scholar with the associated neurocognitive dysfunction likely due to both the deficiency of creatine and the accumulation of guanidinoacetate.5Hanna-El-Daher L. Beard E. Henry H. Tenenbaum L. Braissant O. Mild guanidinoacetate increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain cell development.Neurobiol. Dis. 2015; 79: 14-27Crossref PubMed Scopus (28) Google ScholarIn GAMT deficiency, treatment requires life-long high-dose creatine due to the low blood-brain barrier permeability,18Stockler-Ipsiroglu S. van Karnebeek C. Longo N. Korenke G.C. Mercimek-Mahmutoglu S. Marquart I. Barshop B. Grolik C. Schlune A. Angle B. et al.Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring.Mol. Genet. Metab. 2014; 111: 16-25Crossref PubMed Scopus (74) Google Scholar,20Braissant O. Creatine and guanidinoacetate transport at blood-brain and blood-cerebrospinal fluid barriers.J. Inherit. Metab. Dis. 2012; 35: 655-664Crossref PubMed Scopus (64) Google Scholar as endogenous synthesis is not possible. Oral creatine has an unpleasant taste, making it at times difficult to administer to children. In addition, high-dose creatine administration is not always benign, having resulted in nephrolithiasis in some creatine-deficient patients.21Battini R. Alessandri M.G. Casalini C. Casarano M. Tosetti M. Cioni G. Fifteen-year follow-up of Italian families affected by arginine glycine amidinotransferase deficiency.Orphanet J. Rare Dis. 2017; 12: 21Crossref PubMed Scopus (4) Google Scholar With creatine supplementation, however, GAA still accumulates from peripheral excess,5Hanna-El-Daher L. Beard E. Henry H. Tenenbaum L. Braissant O. Mild guanidinoacetate increase under partial guanidinoacetate methyltransferase deficiency strongly affects brain cell development.Neurobiol. Dis. 2015; 79: 14-27Crossref PubMed Scopus (28) Google Scholar,20Braissant O. Creatine and guanidinoacetate transport at blood-brain and blood-cerebrospinal fluid barriers.J. Inherit. Metab. Dis. 2012; 35: 655-664Crossref PubMed Scopus (64) Google Scholar and while GAA-lowering strategies (e.g., ornithine supplementation, arginine restriction,17Schulze A. Ebinger F. Rating D. Mayatepek E. Improving treatment of guanidinoacetate methyltransferase deficiency: reduction of guanidinoacetic acid in body fluids by arginine restriction and ornithine supplementation.Mol. Genet. Metab. 2001; 74: 413-419Crossref PubMed Scopus (123) Google Scholar which can be difficult to maintain15Mercimek-Mahmutoglu S. Ndika J. Kanhai W. de Villemeur T.B. Cheillan D. Christensen E. Dorison N. Hannig Y. Hofstede F.C. Lion-Francois L. et al.Thirteen new patients with guanidinoacetate methyltransferase deficiency and functional characterization of nineteen novel missense variants in the GAMT gene.Hum. Mutat. 2014; 35: 462-469Crossref PubMed Scopus (26) Google Scholar) can greatly decrease plasma and CSF GAA, brain levels can remain 10 times above normal levels.18Stockler-Ipsiroglu S. van Karnebeek C. Longo N. Korenke G.C. Mercimek-Mahmutoglu S. Marquart I. Barshop B. Grolik C. Schlune A. Angle B. et al.Guanidinoacetate methyltransferase (GAMT) deficiency: outcomes in 48 individuals and recommendations for diagnosis, treatment and monitoring.Mol. Genet. Metab. 2014; 111: 16-25Crossref PubMed Scopus (74) Google Scholar This leaves children at risk for seizures and progressive CNS injury due to the neurotoxicity of GAA.22Hirayasu Y. Morimoto K. Otsuki S. Increase of methylguanidine and guanidinoacetic acid in the brain of amygdala-kindled rats.Epilepsia. 1991; 32: 761-766Crossref PubMed Scopus (18) Google ScholarHere, we describe studies developing a gene therapy approach for GAMT deficiency to overcome the limitations of oral creatine therapy. Restored hepatic gene expression led to weight gain, normalization of plasma and urine GAA levels, restoration of brain and plasma creatine, and resolution of behavioral abnormalities when administered to a murine model of the disorder. These findings have implications for the development of a new therapeutic approach for GAMT deficiency.ResultsAAV-GAMT results in control of GAA and restoration of creatine with a dose-dependent effectTransgenic mice deficient in Gamt were developed as a knockout model and biochemically replicate GAMT deficiency in patients with markedly elevated guanidinoacetic acid and markedly reduced creatine in plasma and tissues.23Schmidt A. Marescau B. Boehm E.A. Renema W.K. Peco R. Das A. Steinfeld R. Chas S. Wallis J. Davidoff M. et al.Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.Hum. Mol. Genet. 2004; 13: 905-921Crossref PubMed Scopus (122) Google Scholar While mice are biochemically similar to human patients, few behavioral deficits have been found.24Torremans A. Marescau B. Possemiers I. Van Dam D. D'Hooge R. Isbrandt D. De Deyn P.P. Biochemical and behavioural phenotyping of a mouse model for GAMT deficiency.J. Neurol. Sci. 2005; 231: 49-55Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Gamt-deficient mice (8 weeks of age, C57Bl/6 background) were administered one of four escalating doses of adeno-associated virus (AAV) expressing human codon-optimized GAMT (hcoGAMT) under a liver-specific (thyroxine-binding globulin [TBG]) promoter to determine the optimal dose for long-term testing (n = 5 per group). Doses of 5 × 1012, 1 × 1013, 5 × 1013, and 1 × 1014 genome copies per kilogram (GCs/kg) were intravenously administered after baseline blood sampling. Mice were euthanized 30 days after administration to assess dose and effect by multiple parameters.AAV vector copy number per diploid hepatic genome in these Gamt−/− mice was determined at each dose (Figure 1A ). As expected, AAV copy numbers increased with increasing dose (mean copies per diploid hepatic genome ±standard deviation [SD]): 5 × 1012 GC/kg: 0.37 ± 0.35; 1 × 1013 GC/kg: 1.58 ± 1.2; 5 × 1013 GC/kg: 33.98 ± 18.28; and 1 × 1014 GC/kg: 75.32 ± 28.60 copy numbers per diploid hepatic genome (n = 5 for 5 × 1012, n = 4 for 1 × 1013, n = 4 for 5 × 1013, and n = 3 for 1 × 1014). Human codon-optimized GAMT RNA was quantified as fold change in gene expression by real-time PCR (n = 5 per dose) (Figure 1B). AAV-mediated hepatocyte GAMT RNA increased with increasing dose (mean ± SD): 5 × 1012 GC/kg: 6,818 ± 4,018; 1 × 1013 GC/kg: 12,058 ± 8,259; 5 × 1013 GC/kg: 41,428 ± 11,590; and 1 × 1014 GC/kg: 69,709 ± 27,152. GAMT protein was examined by western blot, with β-actin as an internal housekeeping protein to evaluate loading. Incremental increases in band density for GAMT were detected with increasing dose (Figure 1C). With quantitation (n = 3 per group), increased protein expression was objectively determined relative to β-actin: 5 × 1012: 0.32 ± 0.17; 1 × 1013: 0.56 ± 0.46; 5 × 1013: 1.11 ± 0.08; and 1 × 1014: 1.63 ± 0.24 (Figure 1D).AAV-mediated liver-specific GAMT expression was also evaluated by in situ hybridization with a codon-optimized human GAMT-specific probe in Gamt−/− mice. With increasing administered AAV doses, hepatic codon-optimized GAMT RNA expression increases, as demonstrated by the greater density of probe-specific chromogenic deposition (red; representative images in Figure 2: A, wild-type [WT] Gamt+/+; B, 5 × 1012 GC/kg; C, 1 × 1013 GC/kg; D, 5 × 1013 GC/kg; E, 1 × 1014 GC/kg). Similarly, hepatic GAMT protein expression (in these Gamt−/− mice) increased with escalating doses as demonstrated by human GAMT-specific immunohistochemistry (representative images in Figure 2: F, WT Gamt+/+; G, 5 × 1012 GC/kg; H, 1 × 1013 GC/kg; I, 5 × 1013 GC/kg; J, 1 × 1014 GC/kg).Figure 2Increasing AAV dose results in augmented GAMT expression and biochemical responseShow full captionAdult Gamt−/− mice were administered AAVrh10 expressing human codon-optimized GAMT intravenously and analyzed 30 days after the injection. RNA expression (A–E) by in situ hybridization (RNAscope) is demonstrated by increasing red precipitate in these representative images (A, untreated; B, 5 × 1012 GC/kg; C, 1 × 1013 GC/kg; D, 5 × 1013 GC/kg; E, 1 × 1014 GC/kg). Protein expression simultaneously increases as detected by immunohistochemical detection and DAB staining (F–J) in these representative images (F, untreated; G, 5 × 1012 GC/kg; H, 1 × 1013 GC/kg; I, 5 × 1013 GC/kg; J, 1 × 1014 GC/kg). With increasing doses, the plasma biochemical response to hepatic hcoGAMT expression increases; plasma creatine incrementally increases and normalizes (K) as elevated plasma guanidinoacetic acid levels abate (L). Values represent means ± standard deviations. (F) and (L) n = 5 per group. Red represents baseline levels in Gamt−/− mice. Blue represents plasma values at 30 days after AAV administration. Black dotted line demonstrates mean plasma creatine level (with range of minimum and maximum represented with gray dotted line) in (K) and mean plasma guanidinoacetic acid level in (L). GAA, guanidinoacetic acid. Bar size in (F) and (G), 100 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)With the marked increase in GAMT hepatic protein, there is an improvement in the metabolic response. While plasma creatine (Figure 2K) did increase (96.38 ± 84.8 nmol/mL) with the lowest dose of vector (5 × 1012 GC/kg) from the pretreatment level (35.53 ± 13.75 nmol/mL [p = 0.013 versus Gamt+/+]), plasma creatine continued to increase sequentially with each dose escalation, becoming equivalent (p = 0.997) to WT plasma creatine levels (249.39 ± 37.6 nmol/mL) at the highest dose (1 × 1014 GC/kg: 237.57 ± 37.4 nmol/mL). Lower doses of vector also resulted in improved plasma creatine levels (1 × 1013 GC/kg: 148.54 ± 96.60, p = 0.13 versus Gamt+/+; 5 × 1013 GC/kg: 196.13 ± 62.10, p = 0.60 versus Gamt+/+) (Figure 2K; n = 5 per group). High-level antibodies to AAV serotype rh10 were present as expected when plasma was tested at 4 months after vector administration (data not shown).Simultaneous with increases in creatine, plasma GAA levels declined. While markedly elevated pretreatment (red data points), a steady decline was detected with incremental dose increases (blue data points) (5 × 1012 GC/kg: 101.55 ± 13.70 versusversus 128.36 ± 22.00 nmol/mL pretreatment, p < 0.0001 versus WT; 1 × 1013 GC/kg: 82.83 ± 29.00 versus 128.69 ± 30.10 nmol/mL pretreatment, p < 0.0001 versus WT; 5 × 1013 GC/kg: 19.512 ± 7.90 versus 119.45 ± 19.00 nmol/mL pretreatment, p = 0.43 versus WT; 1 × 1014 GC/kg: 11.37 ± 0.97 versus 126.37 ± 5.8 nmol/mL pretreatment, expressed as treated versus untreated, n = 5 per group) (Figure 2L). With a dose of 1 × 1014 GC/kg, near equivalency (p = 0.92) to WT (Gamt+/+) mouse plasma levels of GAA (5.17 ± 0.52 nmol/mL) were achieved. As the dose of 1 × 1014 GC/kg achieved the restoration of plasma creatine levels and amelioration of elevated plasma GAA, this dose was chosen for administration and assessment in long-term studies.A single intravenous dose of AAV expressing human codon-optimized GAMT results in improved weight gainWith the optimal intravenous dose now determined, equal numbers of genders and groups (Gamt+/+, Gamt−/−, treated Gamt−/−) of 2-month-old mice were analyzed (n = 8 per genotype group, with 4 males and 4 females included in each group) in a 12-month study. Gamt+/+ and untreated Gamt−/− mice received vehicle alone, while the experimental Gamt−/− group received 1 × 1014 GC/kg of AAV-TBG-hcoGAMT intravenously after baseline blood sampling. Mice were followed for 1 year, with all groups having 100% survival (data not shown). While all male groups started with similar weights (time 0: Gamt+/+ 26.18 ± 1.10 g; Gamt−/− 24.85 ± 1.03 g; treated Gamt−/− 24.53 ± 0.75 g; WT versus treated mutant p = 0.091, WT versus untreated mutant p = 0.214) (Figure 3A ), male mice demonstrated a dichotomy in response: untreated Gamt−/− mice (red data points) had little change in weight after week 10, while WT mice (black data points) continually gained weight throughout the period of study (week 54: untreated Gamt−/− 26.80 ± 1.02 g; Gamt+/+ 38.23 ± 4.53; treated Gamt−/− 32.83 ± 0.83). Overall, by 1 year, AAV-TBG-treated Gamt−/− male mice also demonstrated substantial weight gain (blue data points; 32.83 ± 0.83 g; p = 0.16 versus Gamt+/+) when compared with untreated Gamt−/− mice (26.80 ± 1.02 g; p = 0.02 versus WT), but not to the same extent of the WT controls (38.23 ± 4.53 g).Figure 3GAMT expression by AAV promotes marked improvement in weight and adipose depositsShow full captionMice were weighed weekly from before treatment (week 0) to end of study (week 54). While male AAV-treated Gamt−/− mice gain weight (A, blue line), female mice (B, blue line) within several weeks equal that of the Gamt+/+ (wild type [WT]) littermates (black line); untreated Gamt−/− mice (red line) of both genders were not seen to substantially gain weight after 10 weeks in the study. (C) In these representative images, a female AAV-treated Gamt−/− mouse (right) is of similar size and appearance when compared to a WT littermate (center), while an untreated Gamt−/− mouse remains smaller (left). (Image is from week 54.) (D) Representative microCT images of body fat (brown) in WT mice (Gamt+/+, left), untreated GAMT-deficient mice (center), and GAMT-deficient mice with gene therapy (right). Quantification of weight (E) and body fat amount (F) by microCT at 8 months of age with imaging of Gamt+/+ (WT), Gamt−/−, and Gamt-deficient mice treated with gene therapy. (D–F) Correlation between body weight and body fat in (G) female mice, (H) male mice, and (I) mice of both sexes, determined by the linear regression method using GraphPad Prism 8.0.1. Values represent means ± standard deviations. (A) and (B) n = 4 per group; red represents Gamt−/− mice; blue represents treated Gamt−/− mice; black represents WT controls. (E)–(I) n = 4 per group, except untreated males, where n = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Female AAV-TBG-treated Gamt−/− mice fared even better. Female mice were of a similar weight at the beginning of the study (WT versus treated mutant p = 0.603; WT versus untreated mutant p = 0.338). Similar to the male cohort, untreated Gamt−/− mice (red data points) had little weight gain after week 10 (weight at beginning of study: untreated Gamt−/− 19.08 ± 1.25 g versus 20.23 ± 1.24 g at week 54). AAV-TBG-treated Gamt−/− mice (blue data points) were nearly equivalent in their course of weight gain and weights at 1 year to WT controls (black data points) (at week 54, Gamt+/+ 25.70 ± 1.45 versus 24.95 ± 0.58 g in treated Gamt−/−) (Figures 3B and 3C). Overall, by 1 year, AAV-treated Gamt−/− female mice had substantial weight gain and were comparable to WT mice (p = 0.58), unlike untreated Gamt−/− mice (p = 0.002 versus Gamt+/+).Untreated Gamt−/− mice of both genders were visibly thinner and, when handled, demonstrated less subcutaneous adipose tissue. To better understand the
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